Screening of multi-metal tolerant halophilic bacteria for heavy metal remediation

Hundred and twenty eighty halophilic bacterial isolated from the soil and water of Karnataka mangrove regions were examined for multi-metal tolerance. Bacillus pumilus (accession no.MF472596) was found to be tolerant against four toxic heavy metal ions (Cd2+, Cu2+, Fe3+, and Ba2+) up to 1000 ppm each.

Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume Number 10 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.710.238 Screening of Multi-Metal Tolerant Halophilic Bacteria for Heavy Metal Remediation G Divakar, R.S Sameer* and M Bapuji Acharya & BM Reddy college of Pharmacy, Soldevanahalli, Hesaraghatta Bangalore - 560107, Karnataka, India *Corresponding author ABSTRACT Keywords Mangrove, Heavy metals, Multi-metal tolerance, Bioremediation, Halophilic bacteria Article Info Accepted: 15 September 2018 Available Online: 10 October 2018 Hundred and twenty eighty halophilic bacterial isolated from the soil and water of Karnataka mangrove regions were examined for multi-metal tolerance Bacillus pumilus (accession no.MF472596) was found to be tolerant against four toxic heavy metal ions (Cd2+, Cu2+, Fe3+, and Ba2+) up to 1000 ppm each Chemical analysis was carried out by ICP-AES for Ba2+ and AAS for rest of the metal ions The bioremediation efficiency against metals are as follows Fe>Cu>Ba>Cd (90%, 71%, 52% and 19% respectively) at pH Altering the pH in a range of to10 the bioremediation rate increased to 96%, 88%, 54% and 52% for Fe, Cu, Ba, and Cd respectively The metal absorption efficiency increases on altering pH i.e from 136 ppm, 52 ppm and 35 ppm to 196 ppm, 82 ppm and 60 ppm in Fe3+, Ba2+, Cd2+ respectively, whereas reduction in Cu2+ absorption was noted i.e from 114 ppm to 41 ppm This investigation justifies that specific pH exposure can play a key role in enhancing bioremediation activity of bacterial isolate towards metal ion quality and marketability (Augusto-Costa and Pereira-Duta, 2001) Introduction Industrial activities release many toxic metals to the environment, many of these pollutants are not easily degradable rather persist in environment complicating their remediation These create toxic effects in human (Umrania, 2003), specially when they get accumulated in water bodies available for domestic purpose above the permissible limit (Ba-0.3mg/l, Cu2.0mg/l, Cd-0.003mg/l as per WHO and 0.2mg/l for Fe as per EU) (Lenntech, 2017) This contaminated water interferes with the health and growth of crops, lowering their Remediation and leaching by microbes are gaining attention in the last two decades (Umrania, 2003), as they provide an alternative and eco-friendly method than of the physiochemical techniques in which a huge amount of toxic sludge is left at the end Microbes carry out bio-remediating by three ways: bioaccumulation, biotransformation, and biodegradation (Bestetti et al., 1996) They interact with the metal ion, changing the chemical form by simple oxidation or reduction process (Noghabi et al., 2007; Choi 2062 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 et al., 1996; Ellis et al., 2003) Sometimes the resistant processes are mediated at plasmid level (Zolgharnein et al., 2007) Recent studies have documented the significance of microbe in remediation (Sobhy et al., 2014; Yan and Viraraghavan, 2003; Umrania, 2006; Kozdra and Van Elsas, 2001; Valls and DeLorenzo, 2002; Ajaz et al., 2010) Metal exposure leads to tolerant microbial sp belonging to genera of Bacillus, Corynebacterium, Arthrobacter, Pseudomonas, Ralstonia, Alcaligenes, and Burkholderia (Sobhy et al., 2014; Yan and Viraraghavan, 2003; Umrania, 2006; Kozdra and Van Elsas, 2001; Valls and DeLorenzo, 2002; Ajaz et al., 2010) In our study, we have randomly selected four heavy metal ions of which cadmium belongs to the most toxic group, barium to the minor toxic group whereas iron and copper are essential elements but a higher concentration even of the essential metal in the human body can lead to fatal effects Iron is essential for erythropoiesis as it is the key component of hemoglobin, myoglobin, heme enzyme, metalloflavoprotein and mitochondrial enzyme Overloading of iron in vital organs can lead to cirrhosis, cardiac collapse, cyanosis, metabolic acidosis and pneumoconiosis (Doherty et al., 2006) Even premature death cases and neurogenerative diseases are seen (Atli Arnarson, 2017) Copper has been used for many centuries It is a key component for several metalloenzymes (Kamza and Gitlin, 2002) Its deficiency is uncommon inhuman High concentration of copper intake can cause gastrointestinal distress resulting in, diarrhea, nausea, stomach cramps Injection of a large number of copper salts may produce hepatic necrosis and death (Pizzaro et al., 1999) Barium an alkaline earth metal is relatively abundant in nature High barium doses result in intractable vomiting, severe diarrhea, gastrointestinal hemorrhage and sometimes cardiac arrest leading to death (ASTDR, 2005b) Profound hypoleukemia and muscle weakness leading to flaccid paralysis are an indication of barium poisoning (Johnson and VanTassell, 1991) Cadmium ranks in a close relationship next to lead and mercury as one of the most toxic elements (Jarup et al., 1998) The main source of cadmium is through food for the community, low serum ferritin level in human are noticed for twice the level of cadmium in them (Berglund et al., 1994) once absorbed efficiently retained in human body damaging the kidney, causing chronic pulmonary disease, cardiovascular effects and causing bone demineralization (Bernard, 2008) Cadmium compounds are contemplated to be human carcinogenic (NTP, 2004; Takeuchi, 1977) The present study was carried out with halophilic microbes of Karnataka mangrove region with an objective to search for promising multi-metal resistant halophilic microbes in order to use for remediation from any toxic site from Halophytes of such hypersaline regions are selected for bioremediation due to their metabolic differences than that of terrestrial ones and follow (Oren, 2002; Ventosa et al., 1998; Roberts, 2005; Mevarech et al., 2000; Tehei et al., 2002) they follow compatible solute strategies which can be put into effective use for remediation The isolate was identified as Bacillus pumilus (accession no MF472596) has shown potential for remediation against Cd2+, Cu2+, Fe3+, Ba2+ and was taken up for study in various parameters to get the maximum result 2063 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 Materials and Methods Stock solutions preparation Atomic Absorption Spectrophotometer, SHIMADZU, AA600, was used for bioremediation analysis UV Spectrophotometer, Agilent Technology, Carry 60, was used for spectrophotometric studies Sonicator Probe, Life core, ENUP500, used for sonicating the bacterial cells All media are of Hi-media company and all chemical used are of analytical grade Molecular analysis was carried out in TransDisciplinary University of Health Science and Technology and Eurofins genomics India, Bangalore Stock solutions of Cadmium, Iron, Barium, and Copper (1000 mg/L) were prepared from corresponding metal salts (i.e CdCl2, FeSo4.7H2O, BaCl2.2H2O, CuSO4.5H20) The glassware used for this purpose were leached in 2N HNO3 and rinsed several times with distilled water before use to avoid any metal contamination The Fe2+ is oxidized to Fe3+ in presence of nitric acid 2ltrs of a stock solution of each metal ions was prepared in distilled water and acidified with HNO3 (10-20 ml of 2% HNO3) to prevent precipitation and was sterilized at 121°C for 15 Isolation of bacteria Metal tolerance study of isolates The bacterial strains were isolated from the water and sediment samples, collected during Pre Monsoon (June-July) and Post Monsoon (October-November) season of Mangroves regions from three districts (at twenty different sites) in the Coastal region of Karnataka [i.e Honavar (14.26°-74.44°), Kumta (14.49°74.39°) and Karwar (14.84°-74.11°)] Various concentrations of heavy metals i.e 100-1000 (mg/L) were prepared in a final volume of 10 ml in Hi-media nutrient broth, to which ml of 24 hr old isolated bacterial cultures were inoculated at 37°C for 24 h The tubes were observed for turbidity which was further analyzed by pipetting out 5ml of the sample and analyzing under a UV-spectrum A loopful of the cultures was streaked onto the nutrient agar plate containing respective metal concentration to check for the viability The most potent isolate showing maximum tolerance to the metals was screened by this qualitative method (Pardo et al., 2003) Sediments samples were taken at a depth of 5cm and 40cm from the root region of various trees sp like Sonneratia alba, Kandelia candel, Rhizophora spp, Avicennia spp and water samples were collected at a depth of 30100 cm The samples were incubated in Himedia halophilic broth (M591-500G) for 12 days for enrichment and isolation of extreme halophiles Following ten-fold serial dilution technique in a Hi-media halophilic agar plates, halophilic bacterial isolation was carried out (Rath and Subramanyam, 1996) by incubating o aerobically at 37 C for 48 hours (Das et al., 2012) Pure cultures were obtained by repeated streaking over the nutrient agar plates and preserved in glycerol at -20°C and on nutrient agar slants at 4°C for further use 16S rRNA sequence analysis Bacteria used for our study were preliminarily identified using ABIS online tool based on the cultural, morphological and biochemical characterization Further identification was carried out using 16S rRNA gene sequencing Bacterial genomic DNA was extracted (Peng et al., 2005) The DNA was used as a template for PCR using universal primers These purified products are a template in cycle sequencing (Pitcher et al., 1989) The amplified 16S rRNA gene was purified with 2064 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 QIAGEN Inc kit and electrophoreses on 1% agarose gel Sequencing was carried out in Eurofins (Suganthi et al., 2013; Zhou et al., 1996; Achenbach and Woese, 1995) BLAST program was used to access the DNA similarities and multiple sequence alignment and molecular phylogeny were performed using Bio Edit software Optimization of metal uptake by the isolate Based on the spectrophotometer analysis, the following parameters were chosen for the isolate to be tested under AAS and ICP-AES for metal reduction Remediation of metals by the organisms at pH Optimization of growth parameters Growth characterization Overnight grown bacterial culture in Luria Bertani medium with 5% salt conc was used as inoculums for the analysis of growth pattern It was inoculated in different Erlenmeyer flasks; each containing 100 ml of nutrient broth supplemented with 1000 ppm of different metal solutions incubated at 37°C 5ml of bacterial suspension from each of the flasks was pipetted out after every h and analyzed at 620 nm to monitor the growth pattern (Fig 4) Effect of pH on the isolate Bacillus pumilus was set incubated with varying pH environments (i.e 2, 4, 6, and 10) 5ml of bacterial suspension was pipetted out after every h and analyzed at 620 nm to monitor the growth pattern and tolerance (Fig 5) Effect of pH on metal absorption To check the pH effect on bioremediation, the biomass of Bacillus pumilus was set incubated at different metal concentrations with varying pH environments (i.e 2, 4, 6, 7, and 10) 5ml of bacterial suspension from each of the flasks was pipetted out after the incubation period and analyzed at 620 nm (Silva et al., 2009) (Fig 6) One milliliter of the freshly prepared aliquot of the isolate was incubated in 100 ml of nutrient broth media containing the highest tolerating concentration of respective metal ion CdCl2, FeSO4.7H2O, BaCl2.2H2O, and CuSO4.5H2O The media was adjusted to pH and the cultures were incubated at 37°C for 48 h The incubated cultures were centrifuged at 6500 xg for 20 min, supernatants were used for the determination of the residual metal ion contents by using AAS or ICP-AES (Abou Zeid et al., 2009; Kermani et al., 2010) Controls without inoculation of the bacteria were prepared to detect the initial metal conc Effect of contact time Media containing metal solutions adjusted to pH and inoculated with selected isolate was incubated at 37°C for 72 h The initial and residual conc of metal within the media was measured as mentioned earlier Uptake of metal by the organisms at pH (following cell disruption method) The metal uptake at pH at an optimized temperature and incubation period by the Bacillus sp The cultures were centrifuged at 6500 xg for 20 The pellets were washed with de-ionized water three times and the supernatant was discarded The pellets were sonicated at 70 kHz for 15 at interval and centrifuged at 10000 xg for 20min Bacterial free suspensions were ensured by passing the supernatant through a 2065 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 22µm syringe filter and determined under AAS or ICP-AES for metal uptake (Volesky et al., 1995) containing the same conc of metal, not on spectrophotometric analysis Molecular characterization Effect of highest and lowest pH values As per the spectrophotometer analysis, the highest and lowest pH range which the isolate could tolerate for each metal were selected respectively and observed for bioremediation After 48h incubation the incubated cultures were centrifuged at 6500 xg for 20 The supernatants were used for the determination of the residual metal ion contents by using AAS or ICP-AES (Silva et al., 2009; Abou Zeid et al., 2009) Controls without inoculation of the bacteria were prepared to detect the initial metal conc Uptake of metal by the organisms at highest and lowest pH (following cell disruption method) A comparative study was carried out on the uptake of metal at the highest and lowest pH at an optimized temperature and incubation period The cultures were centrifuged at 6500 xg for 20 The pellets were washed with de-ionized water three times and the supernatant was discarded The pellets were sonicated with 70 kHz for 10mins with interval in between and centrifuged at 10000 xg for 20min The supernatants were passed through a 22µm syringe filter and analyzed under AAS or ICP-AES for metal uptake (Abou Zeid et al., 2009; Kermani et al., 2010; Volesky et al., 1995) For phylogenetic analysis, the 16S rRNA gene sequence of a single band of mw was obtained [Fig (a, b)] This gene sequence, when compared with those retrieved from the GeneBank database, revealed the closest prokaryotic relative of the heavy metal resistant bacteria, KBORMPorg to be Bacillus pumilus in NCBI BLASTN Sequences alignment edition were done using Bioedit (Version 7.2.6) Using the Bootstrap method tree topologies were evaluated in MEGA software providing confidence estimation through phylogenetic tree topologies about the isolate, the sequence was deposited in GenBank under accession number MF472596 (Figure 2) Spectrophotometer analysis of B.pumilus on various metal tolerances the At 620 nm the isolated was analyzed and found that B pumilus can tolerate up to 1000 ppm of all metals (Figure 3) Growth characterization The growth pattern of B pumilus in the presence and absence of metals has been shown in Figure Effect of pH on the isolate The growth pattern and tolerance towards various pH by B pumilus been shown in Figure Results and Discussion Hundred twenty eight halophilic isolates were tested for multi-metal tolerance revealing that cadmium is non-tolerable for the majority of the mangrove isolates Only Bacillus pumilus was found tolerant to all metals was selected for further study The selection is based on the isolate growth on the nutrient agar plate Effect of pH on metal tolerance pH range from 6-10 for the microbe inoculated for 48h is found to be effective in interacting with the metals (Fig 6) 2066 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 Optimization of metal uptake by the isolate Remediation of metals by the organisms at pH Up to 1000 ppm, the isolate had shown tolerance towards Cadmium, Barium, Iron and Copper at pH in 48 h On analyzing with AAS its was found, 90% of metal reduction in the case of Iron, followed by Copper 71%, 50% reduction was found in Barium (ICPAES analyzed) whereas only a 19% reduction was found in the case of Cadmium Cu) pH is the lowest range for the isolate to tolerate Copper, Barium, Iron in which the reduction of metal ranges from 88%, 54%, and 91% respectively pH 10 is the highest for all metals i.e Copper, Iron, Cadmium and Barium in which the metal reduction ranges from 88%, 96%, 52% and 52% respectively pH remain the lowest tolerating range for the isolate in case of Cadmium with a metal reduction of 19% was observed From the above, we can say that pH plays a key role in metal remediation Uptake of metal by the organisms at highest and lowest pH (following cell disruption method) Effect of contact times At pH 7, the isolate was incubated for a period of 72 h with the highest tolerating conc of the Cadmium, Barium, Copper, and Iron to which the reduction was found to be 22%, 52%, 80% and 90% respectively when analyzed under AAS and ICP-AES (only for Barium) There is no effect found in the case of iron and a minimal effect in case of Barium Uptake of metal by the organisms (following cell disruption method) Following the same metal conc (1000 ppm for Cd, Ba, Fe, and Cu) the isolate was grown at pH 7; the cells were disrupted following sonication technique, to detect the uptake of the above metals by the isolate All the filtered supernatant was analyzed in AAS and ICPAES The uptakes of different metal by the isolate are arranged in ascending orders: Cadmium, Barium, Copper, and Iron i.e 35ppm; 52 ppm; 57ppm, 136ppm respectively Effect of highest and lowest pH values Following the spectrophotometer analysis of the isolate for the tolerance of pH at highest and lowest level is considered, it was tested for metal remediation at the same ppm conc as above (i.e 1000 ppm for Cd, Ba, Fe and The isolate grown in the optimized pH was subjected for metal uptake following the above technique The uptake of different metals by the isolate in varying pH subjected for comparison pH is the lowest range for the isolate to tolerate metals like Copper, Barium, and Iron in which the metal uptake ranges from 41 ppm, 82 ppm, and 140 ppm, respectively pH 10 is the highest for all the metals i.e Copper, Iron, Cadmium and Barium in which the metal uptake ranges from 22 ppm, 196 ppm, 60 ppm and 52 ppm respectively pH remains the lowest tolerating range for the isolate in case of Cadmium in which the metal uptake is about 35.1 ppm respectively Bacillus pumilus tolerate all the four heavy metals up to 1000 ppm The resistivity of the microbe towards heavy metals was checked by incubating in different metal solution concentration (Yan and Viraraghavan, 2003; Hall, 1999) The selection procedure of the isolate was based on the growth of bacterial colonies on a nutrient agar plate containing respective metal ions The isolate has shown least tolerance towards Cd, whereas good affinity is observed in the case of Fe, Cu, and Ba The variation in the resistant mechanism 2067 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 of different microbes is the cause of the varying intolerance towards different conc of heavy metals The BLAST hits of KBORMPorg obtained from 16s rRNA gene sequence indicate its close relation with Bacillus pumilus species (accession nos MF472596) Spectrophotometric data reveals the isolate showed a profound growth pattern in the absence of metals except Barium The growth of the isolate can be seen up to 36-40 hr after which it is found to be in standard phase till 48th hr before touching the decline phase The media without metal and Barium supplement, the isolate achieved log phase at a much lower time in comparison to the growth in the presence of other metals Presence of Barium, the growth of the isolate is found to be higher than other The isolate shows highest absorbance value towards all metals in alkaline condition whereas in case of cadmium acidic pH is ineffective, which are taken for effective remediation parameter The evaluation of pH in our work is based on Tehei and Valls conclusion, states the number of cell surface sites available to bind cations, as well as metal speciation, and are affected due to pH variation (Yan and Viraraghavan, 2003) Ajaz and co-workers reported that pH can greatly influence heavy metal removal by microbes (Jalali et al., 2002; Pardo et al., 2003; Hornung et al., 2009; Cappuccino and Natalie, 2002; Pitcher et al., 1989; Acinas et al., 2004; Tamura et al., 2011; Felsenstein, 1985) by influencing the metal speciation and solution chemistry as well as surface properties of bacterial cells The selected isolate subjected to five different parameters for analyzing the remediation of selected heavy metals under AAS as follows; Remediation of metals by the organisms at neutral pH Effect of contact times Uptake of metal by the organisms (analyzed by cell disruption method) Effect of highest and lowest pH values Uptake of metal by the organisms at highest and lowest pH (following cell disruption method) Following Haq et al., AAS and ICP-AES analyzing procedure the selected isolate Bacillus pumilus was prepared by first subjecting it to its highest tolerating conc of the selected heavy metals at pH for a period of 48 h The supernatant was removed at the end of 48 h of the incubation period by centrifugation method and diluted to 1ppm and acidified with HNO3 (Strandberg et al., 1981) The chemical analysis data revealed the removal percentage of each of the heavy metals in descending order Fe>Cu>Ba>Cd, 90%, 71%, 52%, 19%, which made clear about the bioremediation of the metals by the isolate Bacillus pumilus The culture pellets were thus collected and rinsed thrice with PBS and lysed by applying sonication with amplitude of 100 for a period of 20 with 45 sec interval after every 3-4 and acidified with HNO3 and set for AAS analysis The above results corroborate with the work of Haq and co-workers who reported about 86% removal of cadmium (100 mg/l) from medium within 24 h by E Cloacae (Haq et al., 1999) Another report suggests a Cd removal by E Cloacae bacteria isolated from tobacco could reduce only 29% of Cadmium from the medium (Sahoo et al., 2016) 2068 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 Fig.1 Molecular Characterization FIG.1(a) Agarose gel electrophoresis of DNA Sample FIG 1(b) Agarose gel electrophoresis of PCRamplified DNA product Fig.2 Phylogenetic tree based on 16s-rRNA gene partial sequences obtained from the NCBI nucleotide sequence database Fig.3 Spectrophotometer analysis of the B.pumilus on various metal tolerances 2069 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 Fig.4 Growth pattern of B.pumilus Effect of pH on the isolate Fig.5 Growth pattern and pH tolerance of B.pumilus Fig.6 Metal tolerance at different pH and time Effect of pH on metal tolerance 2070 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 Bezverbnaya and Odokuma studied resistant to the heavy metals toxicity by Bacillus sp and Aeromonas sp concluding that the persistence of these isolates in the presence of the respective heavy metals may be as a result of the possession of heavy metal resistant plasmids (Bezverbnaya et al., 2005; Odokuma and Oliwe, 2003) Castillo-Zacarías and coworkers who isolated phenol-resistant bacteria in Monterrey, México from industrial polluted effluents found a Cd2+ removal rate of 23 to 78% by E cloacae, 23 to 64% by P aeruginosa and 24 to 64% by K pneumoniae (Castillo-Zacarías et al., 2011) Kermani and co-workers had reported about cadmium resistant Pseudomonas aeruginosa which tolerate up to a concentration of 80 mg/L (Kermani et al., 2010) Similar results are obtained on Vibrio harvei as studied by (Abd-Elnaby et al., 2011) H Al Daghistani reported four microbial species Bacillus sphaericus, Bacillus pumilus, Panibacillus alvae and G sterothermophilus which have shown a copper remediation of 87.5%, 81%, 65.4% and 79.6% respectively (AlDaghistani, 2012) Shetty and co-workers showed a remediation of 40-70% against copper ion by using Pseudomonas sp (Shetty and Rajkumar, 2009; Vullo et al., 2008; Kumaran et al., 2011) Srikumaran et al., reported a reduction of 62.8% of iron by using a Pseudomonas sp isolated from Uppnar estuarine region (Kumaran et al., 2011) Metal ion binding to the cell surface may be due to covalent bonding, electrostatic interaction, Van-der Waals forces, extracellular precipitation, redox interaction or combination among the processes (Blanco et al., 2000) The negatively charged groups on the bacterial cell wall adsorb metal cations, which retained by mineral nucleation (Wase and Forster, 1997) The contact time between the metal solute and the bacterial cells is an important factor affecting the metal uptake In this study, a minimal change in heavy metal remediation in noticed on increasing the contact time from 48 h to 72 h In the case of Ba, 50% to 52% and Iron from 90.4% to 90.8%, a moderate increase in effect is seen in the case of Cd i.e from 19% to 22% Effective metal remediation was found in case of Cu i.e 71.97% to 88.7% Our result agrees with the results obtained by El-Shanshoury et al., (2012), who had carried the work with B.anthracis (El-Shanshoury et al., 2012) Surface activity and kinetic energy of the solute became more efficient in sorption activity with the rise in temperature, which promote the active uptake or attachment of the metals to the cell surface, respectively (Sag and Kutsai, 2000) Remediation of metals by B pumilus was found to be decreased with increasing temperature above 40°C, which disagree with the results obtained by Mameri and co-worker (Mameri et al., 1999; Prescott et al., 2002; Uslu and Tanyol, 2006) in our case AAS analysis for the sonicated cell for Fe was 136.27 ppm, Cu 114.7 ppm, Ba 52.18 ppm and Cd 35.1 ppm, which clearly confirms the metal absorption capability Babich and Jalali found the pH value as one of the main factors in the bioremediation efficiency and binding to microorganisms (Babich and Stotzky, 1985; Lopez et al., 2000) We have set a highest and lowest pH tolerating level by the isolate towards each metal pH is the lowest range for the isolate to tolerate metals like Copper, Barium, Iron in which the reduction of metal ranges from 88.7%, 54%,91.2% respectively pH remain the lowest tolerating range for the isolate towards Cadmium in which the reduction was found at 19% pH 10 is the highest for all metals i.e Copper, Iron, Cadmium and Barium in which the 2071 Int.J.Curr.Microbiol.App.Sci (2018) 7(10): 2062-2076 metal reduction ranges from 88%,96.2%, 52% and 52% respectively The absorption of four metal ions by B.pumilus at their lowest pH tolerance ability is as follows Fe>Ba>Cu>Cd, with uptake values of 140.23 ppm, 82.31 ppm, 41.79 ppm and 35.1 ppm respectively Similarly, at the absorption of heavy metals at their highest pH tolerance ability, is as follows Fe>Cd>Ba>Cu, with different uptake values of 196.21 ppm, 60.3 ppm,52.18 ppm and 22.13 ppm respectively pH variation plays a critical role in the remediation of metals from the respective solutions An increase in remediation percentage was noticed in all the cases The uptake of the metals like Cd and Fe by the isolate has increased when subjected to an alkaline pH, the Barium uptake is higher in acidic pH whereas Cu removal by the isolate has increased with pH alteration but the metal uptake was greatly affected i.e 114.7 ppm (pH 7) to 22.13 ppm (pH 10) From the results, it could be concluded that the bacterial flora isolated possessed potential in respect of bio-remediation activity The isolate tolerate for several metals, which could be exploited at mining sites or industrial wastewater contaminated regions where the metals are present either individually or in combination under various physiochemical parameter A rise in temperature was ineffective in metal remediation but increasing the contact time has given positive effect in bioremediation of heavy metals The study suggested that altering the pH plays a major role not only in sorption potential of the bacterium but also bio-absorption capacity has increased towards Fe and Cd The multi-metal tolerating Bacillus sp thus appeared to be a suitable candidate in an ecofriendly method for heavy metal ion removal from the environment So, further exploration of the mangrove regions seems effective in finding multi-metal resistant or tolerating microbial species Acknowledgment The work was funded by RGUHS, grant no RGU: R & D: Res Wing 2014-15, dated 13/03/15.-the management of AIT for the facilities, Dr P Mesta, Marine Biologist of IISc field station at Kumta and his team, for their support during fieldwork Dr S P Balasubramani, Molecular biologist of Trans Disciplinary University, Bangalore, for helping us in the identification of isolates, the author is greatly indebted to his Mother who was always with him in his efforts The authors have no conflict of interest 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Oren, 1998 A biology of moderately halophilic aerobic bacteria Microbiol... selected heavy metals under AAS as follows; Remediation of metals by the organisms at neutral pH Effect of contact times Uptake of metal by the organisms (analyzed by cell disruption method) Effect of